Natural plant enzyme inhibitors. Characterization of an unusual alpha-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.).
An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.
D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003512:Cyclohexanones / Q000494:pharmacology; D002523:Edible Grain / Q000201:enzymology*; D004586:Electrophoresis; D008897:Panicum / Q000201:enzymology*; D010434:Pepsin A / Q000494:pharmacology; D010940:Plant Proteins; D011402:Pronase / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000302:isolation & purification*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D003512:Cyclohexanones; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C018031:1,2-cyclohexanedione; D014302:Trinitrobenzenesulfonic Acid; D000516:alpha-Amylases; D010434:Pepsin A; D011402:Pronase